This application describes research designed to determine the biochemical significance of the N-methylation of calmodulin. Calmodulin is a calcium-binding protein and also an important regulator of many calcium-dependent enzymes, particularly those enzymes which underwrite phosphorylation and dephosphorylation reactions. Calmodulin is structurally characterized by the presence of a single residue of trimethyllysine; the biological significance of this post-translational modification is poorly understood, although we have begun to make inroads in this area. Five specific aims define this proposal. The first of these is the purification and characterization of calmodulin N- methyltransferase, which will be accomplished by a number of sequential chromatographic procedures, including fast protein liquid chromatography (FPLC) and affinity chromatography. The second aim is to clarify the biochemical significance of the N- methylation of calmodulin. To achieve this aim we will use purified calmodulin N-methyltransferase to methylated des (methyl)calmodulin and then compare the activities of methylated and non-methylated calmodulins with respect to the activation of several calmodulin-dependent enzymes. The third aim is to determine the relative abundance of non-N-methylated calmodulin in rat tissues (we have prior evidence for the presence of des(methyl)calmodulin). The two calmodulins will be separated and quantitated by sequential melittin-Sepharose chromatography and ion-exchange FPLC. The fourth aim is a comparison of the binding of calmodulin and des(methyl)calmodulin to target proteins in rat tissues. This will be done by iodinating both calmodulins and using them as probes on gel overlay assays. The final aim is to determine those factors which regulate the activity of calmodulin N-methyltransferase. The activity of this enzyme is markedly elevated in liver tumors and in fetal tissues; the difference between enzyme activity in normal and tumor tissue is much greater for this enzyme than for calmodulin itself. The available evidence is that calmodulin is the only substrate for calmodulin N-methyltransferase and we have placed a high priority to determining the significance of the elevation of enzyme activity in rapidly proliferating cells. This research is relevant to basic biochemical processes in cancer and normal developmental processes.